This invention relates to an analytical method in which an analyte and a reactant that reacts with said analyte either directly or indirectly are allowed to react on the analytical areas of a substrate, with the resulting signals being detected for qualitative or quantitative analysis of the analyte, and in which the signals derived from the reaction on the analytical areas of the substrate are detected more intensely than those derived from the non-analytical areas, thereby allowing for higher precision in the analysis of the analyte. More specifically, the invention relates to an analytical method that employs chemical sensors, biosensors such as enzyme sensors, specific binding sensors and which enables precise analysis with a simple sensor configuration by such means as supporting the analyte on projecting analytical areas of the substrate. The invention also relates to an analytical method that enables the fabrication of miniaturized and highly precise microsensors.
Column chromatography, enzyme-chemical analyses, immunoassays and other conventional analytical methods that determine the quantities of target compounds in liquid or gaseous phase have disadvantages such as the need to use large sample volumes for analysis, the need for large analytical equipment and the prolonged time of analysis. These problems present a serious obstacle when there is a need to analyze a large number of samples simultaneously for a single analyte (i.e., simultaneous analysis of multiple samples) or when it is necessary to analyze single samples for a number of different analytical items (i.e., simultaneous analysis for multiple items).
Sensor technology has recently seen marked advances in such applications as chemical sensors, biosensors and specific binding sensors and active efforts are being made toward simplified analytical techniques and miniaturized sensing devices. However, the results are not completely satisfactory and technology is yet to be developed that uses a miniature sensor and which not only enables simultaneous analysis of multiple samples or simultaneous analysis for multiple items but also achieves high precision in these analyses.
To take one example, S. P. Fodor et al. described in Science, Vol. 251, p. 767-773 (1991) a method in which a photolithographic technique was combined with photo-sensitive protective groups to synthesize for analytical use different sequences of peptides or oligonucleotides in minute multiple regions (forming a matrix on a two-dimensional plane). P. Connolly wrote a review article in Trends Biotechnol., Vol. 12, p. 123-127 (1994) to describe a photofabrication process in which a lift-off technique was used to form patterns of hydrophilic and hydrophobic regions in the surface of a substrate. Similar surface processing technologies and analytical methods have been reported by C. R. Lowe et al. (U.S. Pat. No. 4,562,157), S. Nakamoto et al. (Sensors and Actuators, Vol. 13, 165 to 172 (1988), C. S. Dulcey et al. (Science, Vol. 252, 551 to 554, 1991) and S. K. Bhatia et al. Anal. Biochem., Vol. 208, 197 to 205 (1993)!.
W. T. Muller et al. described in Science, Vol. 268, p. 272-273 (1995) a process in which surface functional groups in a mono-molecular layer self-associated onto a substrate were subjected to micro-processing with a scanning probe unit such that a substance could be covalently bonded to minute regions of the substrate.
Surface processing technologies using a scanning tunnel microscope (STM) were also reported by Y. Utsugi NATURE, Vol. 347, 747 to 749 (1990)! and P. Connolly Nanotechnology, Vol. 2, 160 to 163 (1991)!.
D. J. Pritchard et al. reported in Anal. Chim. Acta., Vol. 310, p. 251-256 (1995) a process for the fabrication of a specific binding sensor for simultaneous analysis for multiple items, which comprised reacting photo-sensitive photobiotin with each of two antibodies, with a photomask being applied to a plurality of avidin-immobilized gold electrodes on a silicon wafer substrate.
The above-mentioned micro-processing technologies are all capable of immobilizing different substances onto specified regions of a substrate. However, the fabrication process employed in these technologies includes at least several steps and hence is complicated. In addition, despite the need to process minute regions, expensive reagents such as specific binding substances (antibodies) or precious molecular recognition elements, both of which will determine the characteristics of individual sensing portions presuppose a reaction over the entire surface of the substrate, which makes the conventional technologies not always economical. What is more, the detecting regions such as electrodes must be in correct registry with the immobilized regions and, hence, a highly precise positioning technology is indispensable to the fabrication of miniature sensors. As a further problem, electrodes and other detecting portions will detect not only signals originating from the desired specific binding but also those which derive from undesired events such as the surrounding non-specific binding on the same plane and this makes the conventional technologies unsuitable for precise analyses.
In performing analyses with sensors represented by chemical sensors, biosensors such as enzyme sensors and specific binding sensors such as immunological sensors two functional parts are necessary, i.e., an analyzing part for supporting analytical reagent components such as chemical sensitive substances, bio-catalytic substances, molecular recognition elements and specific binding substances, and a detecting part for detecting signals that are generated as the result of participation of the supported substance. For achieving better precision in analysis, the precision in the amount of the substance to be supported in the analyzing part and the precision of signal detection by the detecting part must both be improved. The precision in the amount of the substance to be supported in the analyzing part depends on various factors such as the method of supporting (e.g. in a free state without being bound chemically, via covalent bonding, via non-covalent bonding, or via a specific binding substance), the precision in the quantity of reaction solution used for supporting, the precision in the quantity of the fluid to be spotted and the precision in the supporting area of the analyzing part. It should particularly be noted here that if the size of the analyzer is reduced to enable analysis of trace amount of samples, it becomes difficult to guarantee the precision in the volume of reaction solution or in the supporting area of the analyzing part. Methods so far adopted to solve these problems comprise providing minute regions of a substrate with a substance binding ability by a photolithographic technique and then bringing the entire surface of the substrate into contact with the substance to be supported. However, this approach has several disadvantages; first, the fabrication process is complicated; second, excess amounts of reagents have to be supported, which makes the approach uneconomical; third, there are unavoidable influences of non-specific adsorption onto regions where the substance of interest should not be supported or those regions to which the substance should not bind.